Poster Presentation 14th Australian Peptide Conference 2022

Rational design of an A3B3 hetero hexameric alpha helical barrel (#114)

Joel J Chubb 1 , Katherine I Albanese 1 , Alison Rodger 2 , Dek N Woolfson 1
  1. University of Bristol, Bristol, BRISTOL, United Kingdom
  2. Molecular Sciences, Macquarie University, Sydney, New South Wales, Australia

Alpha-helical barrels have alpha-helices assembled around a central-axis to generate a solvent-accessible channel1. The channels can be functionalised to introduce small-molecule binding2, transport3, and catalysis4. So far, most alpha-helical barrels reported are homomeric, limiting their functionalisation as features on one helix are repeated in every helix of the scaffold, which can reduce stability or introduce unwanted structural changes, particularly when adding large aromatic side chains5. Here we describe a heteromeric alpha-helical barrel with two different helical chains, A and B.

An A3B3 complex was designed rationally and characterised in solution by: circular dichroism spectroscopy to monitor helicity and thermal stability; analytical ultracentrifugation to determine oligomerisation state; and fluorescence spectroscopy to test for binding of dyes in the channel. In detail, the design of A and B peptides combined known rules for hexameric alpha-helical bundles, and charge complementarity for hetero specification. The individual peptides were unfolded in aqueous solution but associate to form an alpha-helical, hexameric, and highly thermostable complex when mixed. A Job plot confirmed the 1:1 binding stoichiometry and thus formation of the targeted A3B3 complex in solution. Further, the recruitment of a hydrophobic dye confirmed the presence of a solvent-accessible channel. Finally, an X-ray crystallographically derived structure shows good agreement with all solution-phase data. Future work will focus on incorporating metal binding sites within this complex for catalysis.

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